Soybean isolate protein-based composite particle loaded with bovine myocardial peptide and preparation method of soybean isolate protein-based composite particle

Abstract

The invention relates to the technical field of biological embedding, and provides soybean protein isolate based composite particles loaded with bovine myocardial peptide and a preparation method of the soybean protein isolate based composite particles. The preparation method comprises the following steps: fully dissolving soybean protein isolate, bovine myocardial peptide and sodium alginate in water, driving the solution to perform self-assembly by adjusting the pH value of the solution, magnetically stirring, centrifuging, taking supernate, and freeze-drying to obtain the composite particles for packaging bovine myocardial peptide. According to the invention, the soybean protein isolate and the sodium alginate are used as wall materials, and the proportion of embedding materials and packaging conditions are optimized, so that the degradation rate of the bovine myocardial peptide in the body is obviously relieved, the oxidation resistance of the bovine myocardial peptide is effectively retained, the bioavailability of the bovine myocardial peptide is improved, and the bovine myocardial peptide can be used in multiple fields of food, medicine and the like.

Description

Technical Field

[0001] This invention mainly relates to the field of bio-encapsulation technology. Specifically, it selects bovine cardiac muscle peptide as the core active ingredient, uses soy protein isolate and sodium alginate as the main wall materials, and drives the self-assembly of the peptide by adjusting the pH of the solution to obtain composite particles encapsulating bovine cardiac muscle peptide. Background Technology

[0002] Bioactive peptides are composed of different amino acids arranged in various ways, possessing multiple functions such as antioxidation, hypoglycemia, and immunomodulation. In recent years, bioactive peptides have been increasingly used in health foods and pharmaceuticals. However, bioactive peptides are easily decomposed during processing, transportation, and in the gastrointestinal environment, which severely limits their application. Therefore, selecting suitable raw materials as wall materials for loading and delivering bioactive peptides can not only mask their unpleasant odors but also improve their stability during passage through the gastrointestinal tract, expanding their application scope in the food and pharmaceutical fields.

[0003] Soy protein isolate, as one of the most common plant proteins, is widely used in the food industry due to its high nutritional value, good biocompatibility, and low cost. The peptide chains of soy protein isolate molecules are rich in hydrophobic amino acid residues, which can promote hydrophobic interactions between soy protein isolate and hydrophobic bioactive molecules. Simultaneously, the presence of charged amino acid residues allows soy protein isolate to bind to bioactive molecules through electrostatic interactions, forming complexes. Based on these characteristics, in recent years, soy protein isolate has been widely used as a carrier for delivering bioactive substances.

[0004] Sodium alginate, a natural linear anionic polysaccharide, possesses advantages such as low cost, high biocompatibility, and relative stability in the environment. It has been classified as a safe food additive by the U.S. Food and Drug Administration (FDA) and is widely used as a functional food ingredient in the development of functional foods. Furthermore, the linear structure of sodium alginate itself can form an ordered network structure with proteins, improving protein stability. Therefore, this invention uses bovine cardiac muscle peptides as the core active ingredient and soy protein isolate and sodium alginate as wall materials. Composite particles are prepared through self-assembly via electrostatic and hydrophobic interactions between the protein and polysaccharide to improve the bioavailability of bovine cardiac muscle peptides. Summary of the Invention

[0005] To address the key technical challenges in the field of bioactive factor encapsulation and delivery, this invention provides a soybean protein isolate-based composite particle loaded with bovine cardiac muscle peptides and its preparation method. The aim is to study a method for delivering bioactive peptides through protein-polysaccharide composite encapsulation, and also to expand the application scope of bioactive peptides in food, medicine, and cosmetics.

[0006] To address the aforementioned technical problems, the present invention provides the following technical solution:

[0007] Dissolve 0.4 g of soy protein isolate and 0.4 g of bovine myocardial peptide in 20 mL of deionized water. Stir continuously at room temperature for 3.0 h, then adjust the pH of the solution to 4.0 for later use. Dissolve 0.2 g of sodium alginate in 20 mL of deionized water until the solution is completely transparent. Slowly add the soy protein isolate-bovine myocardial peptide mixed solution to the sodium alginate solution, adjust the pH of the mixed solution to 3.5, stir continuously at room temperature for 1.0 h, centrifuge at 8000 g for 10 min, collect the supernatant, and freeze-dry the supernatant at 4℃ for 24 h to obtain the soy protein isolate-bovine myocardial peptide-sodium alginate composite granule powder.

[0008] The obtained composite particles were tested for relevant indicators. The zeta potential value was -37.0 mV. After in vitro gastrointestinal digestion, the DPPH free radical scavenging rate of the composite particles was 31.52%, and the iron ion reducing power absorbance value was 0.17. Compared with the control group that used soy protein isolate as the wall material for encapsulation, the co-encapsulation of soy protein isolate and sodium alginate as composite wall materials significantly alleviated the degradation rate of bovine myocardial peptides in vivo and effectively preserved the antioxidant activity of bovine myocardial peptides. This lays a theoretical foundation for the widespread application of bioactive peptides in many fields such as medicine, functional food and skin care products. Attached Figure Description

[0009] Appendix Figure 1 The graph shows the effect of pH value on the encapsulation efficiency of the composite particles obtained in Example 1 of this invention.

[0010] Appendix Figure 2 The graph shows the effect of the amount of soybean protein isolate added on the encapsulation rate of the composite particles obtained in Example 2 of the present invention.

[0011] Appendix Figure 3 The graph shows the effect of pH value of the soybean protein isolate-sodium alginate mixed solution on the encapsulation rate of the composite particles obtained in Example 3 of the present invention.

[0012] Appendix Figure 4 This is a graph showing the effect of heating temperature on the encapsulation efficiency of composite particles obtained in Example 4 of the present invention.

[0013] Appendix Figure 5 This is a graph showing the Zeta potential results of the composite particles obtained in Example 5 of the present invention.

[0014] Appendix Figure 6 This is a graph showing the differential scanning calorimetry results of the composite particles obtained in Example 5 of the present invention.

[0015] Appendix Figure 7 This is a graph showing the DPPH free radical scavenging rate of the composite particles after in vitro gastrointestinal digestion obtained in Example 5 of the present invention.

[0016] Appendix Figure 8 This is a diagram showing the reduction power of iron ions in the composite particles after in vitro gastrointestinal digestion obtained in Example 5 of the present invention. Detailed Implementation

[0017] Example 1 (1) Dissolve 0.4 g soy protein isolate and 0.4 g bovine myocardial peptide (prepared in the laboratory) in 20 mL of deionized water. Stir continuously at room temperature for 3.0 h, and then adjust the pH of the solution to 4.0, 5.0, 6.0, 7.0 and 8.0 respectively. (2) Dissolve 0.4 g sodium alginate in 20 mL of deionized water and stir magnetically until the solution is completely transparent; (3) Slowly add the soybean protein isolate-bovine myocardial peptide mixed solution to the sodium alginate solution, adjust the pH of the mixed solution to 4.0, stir continuously at room temperature for 1.0 h, centrifuge at 8000 g for 10 min and take the supernatant. Place the supernatant at 4℃ for 24 h and then freeze dry to obtain soybean protein isolate-bovine myocardial peptide-sodium alginate composite particle powder.

[0018] (4) Determine the encapsulation efficiency of the prepared composite particles: The encapsulation efficiency of bovine cardiac peptides encapsulated in soy protein isolate-sodium alginate composite particles was determined by ultraviolet absorption spectrometry. A 10.0 mg / mL composite particle sample solution was prepared. 0.75 mL of 5% trichloroacetic acid solution was added to 1.75 mL of the sample solution, and the mixture was allowed to stand at room temperature for 30 min, followed by centrifugation at 6000 r / min for 10 min. Subsequently, 1.0 mL of the supernatant was taken, and 4.0 mL of biuret reagent was added. The mixture was allowed to stand in the dark for 30 min, and the absorbance was measured at 540 nm. The content of free peptides in the sample solution was calculated using a bovine serum albumin standard curve.

[0019]

[0020] In the formula: m is the mass of the free peptide, g; m0 is the mass of the total added peptide, g.

[0021] Example 2 (1) Take 0.0 g, 0.2 g, 0.4 g, 0.6 g, 0.8 g soy protein isolate and 0.4 g bovine myocardial peptide (prepared in the laboratory) respectively and dissolve them in 20 mL of deionized water. Stir continuously at room temperature for 3.0 h and then adjust the pH of the solution to 4.0. (2) Dissolve 0.4 g sodium alginate in 20 mL of deionized water and stir magnetically until the solution is completely transparent; (3) Slowly add the soybean protein isolate-bovine myocardial peptide mixed solution to the sodium alginate solution, adjust the pH of the mixed solution to 4.0, stir continuously at room temperature for 1.0 h, centrifuge at 8000 g for 10 min and take the supernatant. Place the supernatant at 4℃ for 24 h and then freeze dry to obtain soybean protein isolate-bovine myocardial peptide-sodium alginate composite particle powder.

[0022] (4) The encapsulation rate of the prepared composite particles was determined.

[0023] Example 3 (1) Dissolve 0.2 g soy protein isolate and 0.4 g bovine myocardial peptide (prepared in the laboratory) in 20 mL of deionized water, stir continuously at room temperature for 3.0 h, and then adjust the pH of the solution to 4.0; (2) Dissolve 0.4 g sodium alginate in 20 mL of deionized water and stir magnetically until the solution is completely transparent; (3) The mixed solution of soy protein isolate and bovine myocardial peptide was slowly added to the sodium alginate solution. The pH of the mixed solution was adjusted to 3.0, 3.5, 4.0 and 4.5 respectively. The mixture was stirred continuously at room temperature for 1.0 h. The supernatant was collected by centrifugation at 8000 g for 10 min. The supernatant was placed at 4℃ for 24 h and then freeze-dried to obtain the soy protein isolate-bovine myocardial peptide-sodium alginate composite particle powder.

[0024] (4) The encapsulation rate of the prepared composite particles was determined.

[0025] Example 4 (1) Dissolve 0.2 g soy protein isolate and 0.4 g bovine myocardial peptide (prepared in the laboratory) in 20 mL of deionized water. Stir continuously for 3.0 h at different temperatures (room temperature, 40℃, 50℃, 60℃, 70℃), and then adjust the pH of the solution to 4.0. (2) Dissolve 0.4 g sodium alginate in 20 mL of deionized water and stir magnetically until the solution is completely transparent; (3) Slowly add the soybean protein isolate-bovine myocardial peptide mixed solution to the sodium alginate solution, adjust the pH of the mixed solution to 3.5, stir continuously for 1.0 h at the same temperature, centrifuge at 8000 g for 10 min and take the supernatant. Place the supernatant at 4℃ for 24 h and then freeze dry to obtain soybean protein isolate-bovine myocardial peptide-sodium alginate composite particle powder.

[0026] (4) The encapsulation rate of the prepared composite particles was determined.

[0027] Example 5 (1) Take a certain amount of soy protein isolate and 0.4 g of bovine myocardial peptide (prepared in the laboratory) and dissolve them in 20 mL of deionized water. After stirring continuously at room temperature for 3.0 h, adjust the pH of the solution to 4.0. (2) Dissolve a certain amount of sodium alginate in 20 mL of deionized water and stir magnetically until the solution is completely transparent; (3) The soy protein isolate-bovine cardiac peptide mixed solution was slowly added to the sodium alginate solution, the pH of the mixed solution was adjusted to 3.5, and the mixture was stirred continuously at room temperature for 1.0 h. After centrifugation at 8000 g for 10 min, the supernatant was collected. The supernatant was placed at 4℃ for 24 h and then freeze-dried to obtain soy protein isolate-bovine cardiac peptide-sodium alginate composite granule powder. The total weight of soy protein isolate and sodium alginate was 0.3 g, and the mass ratio (SPI / SA) was 1:0, 4:1, 2:1, 1:1, 1:2, 1:4 and 0:1, respectively. The zeta potential and antioxidant properties after in vitro gastrointestinal digestion of the prepared composite granules were determined.

[0028] (4) Zeta potential The zeta potential of dispersions (composite particles with SPI / SA mass ratios of 1:0, 4:1, 2:1, 1:1, 1:2, 1:4, and 0:1) were measured using a Nano Zetasizer analyzer (ZEN3600, Malvern Instruments, UK). All test solutions were diluted to 1.0 mg / mL with deionized water prior to testing. Multiple evaluations of individual samples were performed to minimize experimental error.

[0029] (5) Differential scanning calorimetry (DSC) The thermal properties of soy protein isolate-bovine cardiac peptide-sodium alginate composite particles were measured using a DSC Q2000 instrument. The sample (4.0 ± 0.4 mg) was sealed in an aluminum dish and then heated from 20°C to 300°C at a heating rate of 10°C / min. The sample holder was rinsed with nitrogen at a flow rate of 50 mL / min.

[0030] (6) DPPH free radical scavenging rate Preparation of test sample solution: Add 4.0 mL of the test solution and 0.2 mmol / L DPPH-95% ethanol mixture to each solution and mix well. After standing at room temperature in the dark for 30 min, centrifuge at 6500 g for 10 min and take the supernatant. Detect its absorbance value at a wavelength of 517 nm. Preparation of the control group for the test sample: Take 4.0 mL of the sample solution and mix it with 95% ethanol solution. After treatment under the same conditions, measure its absorbance value. Preparation of blank control samples: Mix 4.0 mL of 0.2 mmol / L DPPH-95% ethanol solution and deionized water, shake well, treat under the same conditions, and then measure the absorbance. The formula for calculating the DPPH free radical scavenging rate is as follows:

[0031] In the formula, A is the absorbance value of the sample solution group to be tested; A1 is the absorbance value of the control group of the sample to be tested; and A0 is the absorbance value of the blank group of the sample to be tested.

[0032] (7) Reducing power of iron ions Add 2.0 mL of phosphate buffer (pH 6.6) and 2.5 mL of 1.0% potassium ferricyanide solution to 1.0 mL of 10 mg / mL sample solution, mix well, and react in a water bath at 50.0℃ for 20 min. After the reaction, cool to room temperature, add 2.5 mL of 10.0% trichloroacetic acid solution, mix well, and centrifuge at 3000 g for 10 min. After centrifugation, take 2.5 mL of the supernatant and add the same volume of deionized water, mix well, add 0.5 mL of 0.1% ferric chloride, react at room temperature for 10 min, and measure the absorbance at 700 nm using a UV spectrophotometer.

[0033] Results and Analysis (1) The results obtained in Example 1 are shown in the appendix. Figure 1 As shown, the encapsulation efficiency of the prepared composite particles for bovine cardiac peptides decreased significantly with increasing pH of the soy protein isolate-bovine cardiac peptide mixed solution. This may be because the isoelectric point of soy protein isolate is around pH 4.5. When the pH is higher than the isoelectric point of the protein, the surface of the soy protein isolate molecules is negatively charged, while sodium alginate itself is a large-molecule anionic polysaccharide. Therefore, there is electrostatic repulsion between the protein and the polysaccharide, resulting in decreased stability of the composite particles and a reduced encapsulation efficiency. When the pH is lower than the isoelectric point of the protein, the surface of the soy protein isolate molecules carries a net positive charge. The soy protein isolate and sodium alginate form a more stable complex through electrostatic interaction, thus resulting in a higher encapsulation efficiency of the composite particles for bovine cardiac peptides.

[0034] (2) The results obtained in Example 2 are shown in the appendix. Figure 2As shown, with the increase of soy protein isolate, the encapsulation efficiency of the prepared composite particles for bovine cardiac peptides showed a trend of first increasing and then decreasing. This may be because at a lower addition amount (0.2 g), soy protein isolate molecules, as the main wall material, form a complex with sodium alginate through electrostatic interaction under pH control, increasing the number of encapsulation sites and significantly improving the encapsulation efficiency compared to the group without added soy protein isolate. However, when the addition amount of soy protein isolate exceeds the critical value, excessive soy protein isolate may cause aggregation between molecules, forming particle precipitation, which not only damages the stability of the composite particles but also reduces their encapsulation efficiency.

[0035] (3) The results obtained in Example 3 are attached. Figure 3 As shown, the encapsulation rate of the composite particles gradually decreases with increasing pH of the soy protein isolate-sodium alginate mixed solution. This is mainly because as the pH of the mixed solution increases, the positive charge of the soy protein isolate gradually decreases, while sodium alginate always carries a negative charge, leading to a gradual weakening of the electrostatic attraction between the soy protein isolate and sodium alginate, resulting in decreased stability of the composite particles and a relatively loose encapsulation structure.

[0036] (4) The results obtained in Example 4 are attached. Figure 4 As shown, the encapsulation efficiency of bovine cardiac peptides by the composite particles did not change significantly as the heating temperature gradually increased from room temperature to 70℃. This result may be because within this temperature range, soy protein isolate did not undergo significant thermal denaturation, its native conformation was maintained, and the electrostatic interaction with sodium alginate and the complex structure remained intact, thus maintaining the integrity of the encapsulation structure. Therefore, unlike factors such as pH and the amount of soy protein isolate added, moderate heat treatment (≤70℃) has a relatively small impact on the encapsulation efficiency of this composite system, which facilitates the use of mild heat sterilization processes in actual processing.

[0037] (5) The zeta potential value is closely related to the surface charge of the molecules and is an important indicator for evaluating the interaction and stability of the system. Compared with the SPI / SA1:0 composite particles, the composite particles with the addition of sodium alginate all exhibited negative potentials. Sodium alginate is a macromolecular anionic polysaccharide. When the amount of sodium alginate added is relatively low, the negatively charged sodium alginate and the positively charged soy protein isolate increase the absolute value of the zeta potential of the composite particles due to electrostatic interaction. However, when the amount of sodium alginate added exceeds a certain range, the sodium alginate in the composite particles provides more negative charge, resulting in higher electronegativity and electrostatic repulsion in the soy protein isolate-bovine myocardial peptide-sodium alginate composite particles. The highly branched neutral sugar side chains on the sodium alginate molecule stabilize the structure, prevent the aggregation of soy protein isolate, and expose more charged amino acid residues (as shown in the appendix). Figure 5 (As shown).

[0038] The thermal stability of proteins in food determines their processing conditions; improving thermal stability can increase the processing range of proteins. (Appendix) Figure 6 The SPI / SA1:0 group exhibited a broad endothermic peak at 91.2℃, primarily due to the glass transition temperature of the protein. With the addition of sodium alginate, the position of this endothermic peak shifted to varying degrees; this result indicates that the hydrophobic and electrostatic interactions between soy protein isolate and sodium alginate enhance the structural and thermal stability of soy protein isolate, resulting in a higher denaturation temperature. Simultaneously, the soy protein isolate-bovine cardiac peptide-sodium alginate composite particles generated an endothermic peak around 200℃ due to intermolecular interactions, indicating a subtle decomposition of the complex at 200℃. The SPI / SA2:1, 1:1, 1:2, 1:4, and 0:1 groups showed an exothermic peak at 240℃, corresponding to the melting process of alginate.

[0039] Appendix Figure 7 and attached Figure 8 The antioxidant properties of the composite particles after in vitro gastrointestinal digestion were analyzed. With increasing sodium alginate content, the DPPH free radical scavenging rate and ferric reducing power of the composite particles after in vitro gastrointestinal digestion showed a trend of first increasing and then decreasing. This result is mainly because, at a fixed total wall material concentration, the mass ratio of soy protein isolate to sodium alginate affects the structure, digestive stability, and release of active substances of the composite particles. Within the optimal mass ratio range (e.g., SPI / SA 2:1 or 1:1), the composite particles maintain moderate stability in the gastric acid environment and slowly release active substances after entering the intestine, which may prolong the antioxidant time of bovine cardiac peptides. When the sodium alginate ratio is too high, the composite particle structure becomes too dense or forms an excessively strong barrier, which inhibits the action of digestive enzymes, leading to incomplete release of active substances. Conversely, when the soy protein isolate ratio is too high, the composite particles degrade prematurely in the stomach, resulting in early release of active substances and a shortened duration of antioxidant activity.

[0040] (6) Overall, the structure of the soy protein isolate-bovine myocardial peptide-sodium alginate composite particles obtained in this invention is relatively stable, and the encapsulation rate of the core active factor bovine myocardial peptide is high. Compared with composite particles encapsulated only with soy protein isolate or sodium alginate, the soy protein isolate-bovine myocardial peptide-sodium alginate composite particles (SPI / SA2:1) have a more stable structure, good thermal properties, and after in vitro gastrointestinal digestion, their DPPH free radical scavenging rate and iron ion reducing power are significantly higher than those of other composite particles, effectively preserving the antioxidant properties of bovine myocardial peptide itself.

Claims

1. A soybean protein isolate-based composite particle loaded with bovine cardiac muscle peptides and its preparation method, characterized in that, Includes the following steps: Take a certain amount of soy protein isolate (SPI) and 0.4 g of bovine myocardial peptide (BMP, prepared in the laboratory) and dissolve them in 20 mL of deionized water. After stirring continuously for 3.0 h, adjust the pH of the solution to the preset range for later use. Dissolve a certain amount of sodium alginate (SA) in 20 mL of deionized water until the solution is completely transparent; The soy protein isolate-bovine cardiac peptide mixed solution was slowly added to the sodium alginate solution. The pH of the mixed solution was adjusted to a suitable range, and the mixture was stirred continuously for 1.0 h. After centrifugation at 8000 g for 10 min, the supernatant was collected. The supernatant was placed at 4℃ for 24 h and then freeze-dried to obtain the soy protein isolate-bovine cardiac peptide-sodium alginate composite granule powder.

2. The method for preparing soybean protein isolate-based composite particles loaded with bovine cardiac muscle peptides according to claim 1, characterized in that, The pH values ​​of the soy protein isolate-bovine cardiac muscle peptide mixed solutions were 4.0, 5.0, 6.0, 7.0, and 8.0, respectively; the soy protein isolate addition amounts were 0.0 g, 0.2 g, 0.4 g, 0.6 g, and 0.8 g, respectively; the pH values ​​of the soy protein isolate-sodium alginate mixed solutions were 3.0, 3.5, 4.0, and 4.5, respectively; and the stirring temperatures were room temperature, 40℃, 50℃, 60℃, and 70℃, respectively. Subsequently, composite particles were prepared under the determined optimal conditions, wherein the total weight of soy protein isolate and sodium alginate was 0.3 g, and the mass ratio (SPI / SA) was 1:0, 4:1, 2:1, 1:1, 1:2, 1:4, and 0:1, respectively.

3. Prepare soybean protein isolate-bovine cardiac peptide-sodium alginate composite particles according to the preparation method described in claims 1 and 2.

4. The soybean protein isolate-based composite particles loaded with bovine cardiac muscle peptides and the preparation method thereof according to claim 3, characterized in that, The encapsulation efficiency of the composite particles was 60.04-71.60%, the absolute value of the Zeta potential was 16.40-43.50 mV, and the DPPH free radical scavenging rate of the composite particles after in vitro gastrointestinal digestion was 28.30-33.57%, and the absorbance value of the iron ion reducing power was 0.13-0.17.

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